In article <CoMBtv.I1M at freenet.carleton.ca>, ar229 at FreeNet.Carleton.CA (Allison Haggarty) writes:
>>> I have been using X-gal to stain cells blue after transient tranfections.
> The cells are blue allright but there are lots of crystals in the stain so
> the results are not very photogenic. The problem seems to arise when the
> X-gal stock (10% in DMF, frozen at -20 C) is diluted 1:100 in the working
> buffer for staining (5mM K ferricynanide, 5mM K ferrocyanide, 2 mM MgCl2,
> made up in PBS). As soon as the X-gal hits the aqueous buffer the
> solution goes cloudy.
> Does anyone know how to prevent this? Am I stuck with filtering?
> Thanks.
>> --
> Allison Haggarty
Hello,
I use a slightly different protocol; 0.1 M phosphate pH 7.4 instead
of PBS, and I include Nonidet NP40 (0.02%) and deoxycholate (0.01%).
I add the X-gal (to 1 mg/ml final) as a 2% stock solution in DMF.
When does the pptn occur? If its just when you make it up then add
the X-gal solution more slowly (maybe use a more dilute stock solution).
If its when it hits the cells then make sure they have been rinsed well
after fixation.
I routinely filter the staining solution (0.45 um filter) when I
make it at first and when stored in the dark it lasts for weeks. When
precipitation does occur then it still works after another round of filtration.
In the protocol I was originally given it was suggested that, after
a suitable level of staining had been achieved, the cells be washed with
phosphate buffer containing 3% DMSO. I've never done this as I usually
photograph the cells first.
I hope it all works out,
Bernard
(What's wrong with filtering, anyway?)
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)